1. Field of the Invention
This invention relates to shaped objects for pyrogen retention, processes for their manufacture, and use thereof.
2. Description of Related Art
It is well known that pyrogens are substances, such as lipopolysaccharide complexes, that in extremely small amounts (approximately 0.2 xcexcg/kg body weight) cause fever in higher-order animals and humans after intravenous injection (Pschyrembel, xe2x80x9cKlinisches Wxc3x6rterbuchxe2x80x9d (Clinical Dictionary), 257th Ed., de Gruyter (1994), p. 1279). For this reason, infusion media contaminated with pyrogens, for example, must be rendered pyrogen-free prior to use.
A further area of application in which the removal of pyrogens is important is kidney dialysis using so-called high-flux membranes. Here, the dialyzate must either be freed of pyrogens, using a separate membrane unit for example, to prevent pyrogens from being filtered back through the wall of the dialysis membrane and thus entering the blood of the dialysis patient, or it is also possible to use a dialysis membrane that itself does not pass pyrogens, thus also preventing the transition of pyrogens into the blood. In this case, the pyrogen retention may be effected by the membrane""s separation characteristic, i.e., the pyrogens are retained due to their size. In many cases, the pyrogen retention is based on adsorptive effects.
EP-A 0 478 842 describes a membrane filter layer made from inert polymer materials such as polyethylene, polypropylene, nylon 6,6, polycaprolactam, polyester, or polyvinylidene fluoride, from which membranes with a pore size of 0.04 to 0.45 xcexcm can be produced for pyrogen removal, whereby the polymer material for the membrane filter layer is preferably a cationically or anionically modified polymer since the deposition capacity can be attained more readily. One example of a cationically modified polymer is nylon 6,6, whose surface is modified with a thermally hardenable polymer having quaternary ammonium groups. For the anionically modified polymers, carboxyl groups are preferred as the source of negative charge.
It is well known that pyrogens include the endotoxins, which represent cell fragments of dead gram-negative bacteria. With respect to endotoxin removal, WO 94/17906 describes a hydrophilic, charge-modified, microporous membrane having a cross-linked structure comprising an interpenetrating polymer network, whereby the membrane comprises polyethersulfone, polyfunctional glycidyl ether, polymeric amine, and possibly a homopolymer made from vinylpyrrolidone or a quaternized copolymer made from vinylpyrrolidone and dimethylaminoethyl methacrylate quaternized with methyl sulfate.
WO 98/01208 describes cationically charged membranes suitable for removing endotoxins. According to this publication, the membranes are manufactured by bringing a hydrophobic polymer membrane, preferably made from polysulfone, polyarylsulfone, or polyethersulfone, into contact with a polymer wetting agent and thereby cross-linking at least one agent cationically modifying the membrane onto the membrane. In another embodiment of WO 98/01208, the membrane is cast from a solution comprising polyethersulfone, a copolymer of vinylpyrrolidone and a cationic imidazolinium compound, preferably methylvinylimidazolinium methyl sulfate, and a low-molecular organic acid, whereby weight percentages for the acid in the casting solution of 24 to 34% by weight are disclosed. For this reason, the system components coming into contact with the casting solution must be acid-resistant, thus making the system more expensive.
The prior art discloses charge-bearing structures for pyrogen or endotoxin removal. Charge-bearing structures are preferred at least in order to attain a retention effect beyond that of the sieve effect. It is often noted, however, that the action of membranes modified in this manner is not sufficient, i.e., the endotoxin retention attained thereby is not high enough. Therefore, a need still exists for a product with improved retention of pyrogens or endotoxins, and for a process for manufacturing such a product.
The present invention is directed to a hydrophilically acting shaped object for pyrogen retention comprising a synthetic polymer component and a copolymer additive made from vinylpyrrolidone and a vinylimidazole compound, and a method of making the same.
Surprisingly, it has been found that, due to the additive of the invention, the shaped object of the invention has an improved endotoxin retention capacity that is higher than that of a comparable charge-modified shaped object.
It is clear that the additive should be accessible to the pyrogens, i.e., present on the surface of the shaped object. Preferably, the additive is therefore either worked into the shaped object homogeneously, or, more preferably, present on the surface of the shaped object in increased concentration.
The shaped object of the invention may be pore-free. In this embodiment, the invention may be, for example, in the form of a correspondingly fine-grained powder used as an adsorption medium for pyrogen retention columns.
Preferably, however, the shaped object of the invention has a porous structure, and it is especially preferred for it to have a porous structure open toward the surface and thus to have a pore surface in addition to the geometrical surface. Such objects have a large surface area comprising the external geometric surface and the inner surface of the pores, whereby the additive is accessible both on the geometric and pore surfaces. Due to its pore size, the shaped object of the invention allows at least part of the pyrogens to pass. The additive of the invention, present on the pore surface, acts to retain these pyrogens.
It is especially preferred for the porous shaped object to be a semipermeable flat, tubular, or hollow-fiber membrane, whereby one skilled in the art will understand how large the dimension of the pores should be in order to maintain the pressure build-up within acceptable limits, yet understand how small the pores should be to maintain sufficient pore surface and thus sufficient load capacity of the membrane for the pyrogens or endotoxins.
The copolymer of the invention may have any known copolymer structure, such as, for example, a block copolymer. Preferably, the copolymer is a statistic copolymer.
In accordance with the invention, the molar ratio of the vinylimidazole compound to the vinylpyrrolidone in the copolymer may be selected from a broad range. Preferably, the ratio is from 10:90 to 90:10 mol/mol. A ratio of about 50:50 mol/mol is especially preferred.
In accordance with the invention, the vinylimidazole compound may be substituted at one or more of carbon atoms 2, 4, and 5, whereby the vinylimidazole compound is preferably 1-vinylimidazole substituted at carbon atoms 2 and/or 4 and/or 5 with a C1-C5-alkyl group. In another preferred embodiment of the invention, the vinylimidazole compound is 1-vinylimidazole.
For many of the applications of the shaped object of the present invention, the adsorptive adhesion of the additive on the synthetic polymer component is sufficient, such that no after-treatment is required to increase the additive adhesion. For other applications of the shaped object of the invention, in which, without after-treatment, a portion of the additive may be eluted (for example by sterilization, extraction, or another after-treatment step of the shaped object such as pre-flushing of the membrane, or by the pyrogen-contaminated liquids used) the shaped object of the invention may contain the additive in a form that sufficiently increases adhesion. Preferably, the additive is cross-linked to increase adhesion.
Since the pyrogen- or endotoxin-containing solutions are generally aqueous, the synthetic polymer component must act hydrophilically, no later than when the shaped object is used according to the invention. To this end, basically any structure is suitable that renders the geometric surface, and the porous surface in the case of a porous shaped object, hydrophilic. For example, the hydrophilic action may be attained by treating the shaped object under pressure with known wetting agents such as ethanol, isopropyl alcohol, or a mixture of ethanol and/or isopropyl alcohol with water, or with water alone.
In the scope of the present invention, however, the following embodiments are preferred, which render the shaped object permanently hydrophilic. In one of these preferred embodiments, the synthetic polymer component is a hydrophilic polymer, whereby the polymer may also be one that was originally hydrophobic and was chemically hydrophilized by, for example, amidation, amination, carboxylation, sulfonation, or hydroxylation. Especially preferred hydrophilic polymers are polyamide 6, polyamide 6,6, polyamide 4,6, polyethylvinyl alcohol, or sulfonated polyethersulfone.
In another preferred embodiments, the synthetic polymer component comprises a mixture of a hydrophobic polymer with a hydrophilic modification of this polymer. In another preferred embodiment of the invention, the synthetic polymer component is a hydrophobic first polymer and a hydrophilic second polymer. In an especially preferred embodiment of the invention, the hydrophobic first polymer is selected from the group comprising polysulfones, polyethersulfones, polyarylethersulfones, polyacrylonitriles, polycarbonates, or polyolefins such as polyethylene or polypropylene. In another especially preferred embodiment of the invention, the hydrophilic second polymer is selected from the group comprising polyvinylpyrrolidones, polyethylene glycols, polyvinyl alcohols, or sulfonated polyethersulfones.
The invention further comprises a first process for manufacturing a hydrophilically acting shaped object for pyrogen adsorption, comprising an additive and a synthetic polymer component, by preparing a solution comprising a synthetic polymer component, shaping the solution, subjecting the solution to phase separation that is thermally induced or induced by a non-solvent, resulting in the shaped object, washing, subsequent drying of the shaped object, and possibly treating with a wetting agent. In this embodiment, a copolymer made from vinylpyrrolidone and a vinylimidazole compound is selected as the additive and the additive is added either to the solution comprising the synthetic polymer component, or the additive is added in a manufacturing step for the shaped object following the shaping of the solution.
The shaped object of the invention may be manufactured pore-free and in this embodiment is suitable, in the form of a correspondingly fine-grained powder for example, for use as an adsorption medium for pyrogen retention columns.
In a preferred embodiment, hydrophilically acting shaped objects manufactured using the process of the invention are semipermeable flat, tubular, or hollow-fiber membranes with porous structure.
In the embodiment of the first process of the invention, in which the additive is added to the solution containing the synthetic polymer component, the additive is preferably added in a ratio of 0.1 to 10% with respect to the weight of the solution. The additive may also be added in cross-linked form, such as in the form of BASF LUVICROSS VI or LUVICROSS VI-M.
In another emnodiment of the first process of the invention, the additive used in manufacturing the shaped object can also be added in a step following the shaping of the solution. For example, the additive may be contained in the coagulant that is transited by the shaped solution.
In another preferred embodiment of the first process of the invention, the additive in manufacturing the shaped object is added in a step following shaping of the solution. The shaped object is a hollow-fiber membrane and the solution containing the synthetic polymer component is shaped via a hollow-fiber nozzle into a hollow fiber and an interior solution containing the additive is fed via the interior bore of the hollow-fiber nozzle. In an especially preferred embodiment of the invention, the interior solution contains the additive in a concentration of 0.1 to 5% by weight.
The shaped object of the invention is manufactured according to a second process of the invention by adding, as an additive to a polymer object made from a synthetic polymer component, a copolymer made from vinylpyrrolidone and a vinylimidazole compound. The polymer object may be produced using a known manufacturing process for polymer objects, for example as a powder, such as by phase separation induced thermally or by non-solvents.
In a preferred embodiment of the second process of the invention, the polymer object is a semipermeable flat, hollow-fiber, or tubular membrane with a porous structure, such as is produced by a process with diffusive or thermal phase separation.
In the second process of the invention, the additive can be added to the polymer object using any of the known application processes such as impregnation, spraying with a solution of the additive, or immersion in a solution containing the additive. In a preferred embodiment of the second process of the invention, the additive is dissolved in a bath and the polymer object drawn through the bath and dried, whereby the solution can be water and/or an organic solvent such as methanol, ethanol, or isopropanol. In an especially preferred embodiment of the second process of the invention, the additive is dissolved in the bath in a concentration of 0.001% to 2.0% by weight. A bath having a concentration of 0.01% to 1.0% by weight is highly preferred.
The copolymer of the invention used as the additive may have any known copolymer structure, such as for example, a block copolymer. Preferably the copolymer is a statistic copolymer.
In accordance with the invention, the molar ratio of the vinylimidazole compound to the vinylpyrrolidone in the copolymer may be selected from a broad range. Preferably, the ratio is from 10:90 to 90:10 mol/mol. A ratio of about 50:50 mol/mol is especially preferred.
In accordance with the invention, the vinylimidazole compound may be substituted at one or more of carbon atoms 2, 4, and 5, whereby the vinylimidazole compound is preferably 1-vinylimidazole substituted at carbon atoms 2 and/or 4 and/or 5 with a C1-C5-alkyl group.
In another preferred embodiment of the invention, the vinylimidazole compound is 1-vinylimidazole.
In order for the shaped object of the invention to act hydrophilically, basically any process is suitable that renders the geometric surface, and the porous surface in the case of a porous shaped object, hydrophilic. The hydrophilic action can be attained by treating the shaped object under pressure with known wetting agents such as ethanol, isopropyl alcohol, or a mixture of ethanol and/or isopropyl alcohol with water, or with water alone. However, the embodiments of the invention described in the following, which render the shaped object permanently hydrophilic, are preferred.
In one of these preferred embodiments, the synthetic polymer component is a hydrophilic polymer. The polymer may also be one that was originally hydrophobic and was chemically hydrophilized by, for example, amidation, amination, carboxylation, sulfonation, or hydroxylation. Especially preferred hydrophilic polymers are polyamide 6, polyamide 6,6, polyamide 4,6, polyethylvinyl alcohol, or sulfonated polyethersulfone.
In another of these preferred embodiments, the synthetic polymer component is a mixture of a hydrophobic polymer with a hydrophilic modification of this polymer. In another of these preferred embodiments of the invention, the synthetic polymer component is a hydrophobic first polymer and a hydrophilic second polymer. In an especially preferred embodiment of the invention, the hydrophobic first polymer is selected from the group comprising polysulfones, polyethersulfones, polyarylethersulfones, polyacrylonitriles, polycarbonates, or polyolefins such as polyethylene or polypropylene. In another especially preferred embodiment of the invention, the hydrophilic second polymer is selected from the group comprising polyvinylpyrrolidones, polyethylene glycols, polyvinyl alcohols, or sulfonated polyethersulfones.
For many applications of the shaped object of the invention, the adsorptive adhesion of the additive on the synthetic polymer component is sufficient, so that no after-treatment is required to increase the additive adhesion for these applications.
For those applications of the shaped object of the invention in which, without after-treatment of the additive, a portion of the additive would be eluted (for example by sterilization, extraction, or another after-treatment step such as pre-flushing of the membrane or by the pyrogen-contaminated liquids used) the adhesion between the additive and the shaped object is sufficiently increased by an after-treatment, whereby the additive is preferably cross-linked. All known processes for cross-linking copolymers made from vinylpyrrolidone and a vinylimidazole compound may be used, for example high-energy irradiation such as xcex3 or UV radiation, with or without UV initiator, or thermal processes with a peroxide initiator.
In the first process of the invention, the additive is preferably added to the coagulant together with a thermal or UV initiator. In the production of hollow-fiber membranes, the additive is preferably added to the interior solution together with a thermal or UV initiator and thermally or photochemically cross-linked in a process step following coagulation, such as during drying.
In the second process of the invention, the additive is preferably pre-placed in a bath together with a UV initiator, and the polymer object is drawn through the bath and after leaving the bath subjected to irradiation with a UV lamp and dried. The shaped object may be pre-dried after leaving the bath and before irradiation.
While the additives commonly used in the art for improving pyrogen retention have always produced a shaped object that is charge-bearing, the additive of the invention results in shaped objects that bear no charge. It must be regarded as surprising that the shaped objects of the invention or those manufactured using the processes of the invention exhibit a significantly improved pyrogen retention compared to shaped objects without this additive, so that, in this manner, liquids originally contaminated with pyrogens have a pyrogen concentration, after treatment with the shaped objects of the invention, that is below the detection limit of conventionally employed test methods.
It is also surprising that the shaped objects of the invention or those produced using the processes of the invention with the vinylpyrrolidone/vinylimidazole copolymer as an additive, which bears no charge, even have significantly higher pyrogen retention than prior-art shaped objects that bear a charge, such as shaped objects with the charge-bearing copolymer made from vinylpyrrolidone and methylvinylimidazolinium methyl sulfate. For this reason, the shaped objects of the invention for retaining pyrogens and in particular endotoxins can be used with particular advantage.
As previously noted and well known, pyrogens include endotoxins. In the following examples, the pyrogen retention capacity is determined based on the example of the endotoxin retention capacity, whereby hollow-fiber and flat membranes are tested. To this end, endotoxin-containing test solutions are filtered through the membranes to be tested, under defined conditions described in more detail in the following, and the endotoxin concentrations in the filtrates are measured in comparison to those in the test solution. On the filtrate side, sterile, pyrogen-free materials are used exclusively. The depyrogenation/sterilization of the stainless-steel filtration equipment and the glass sample vessels is performed by heating for at least 3 hours to 180xc2x0 C. The endotoxin concentration in the filtrates and test solutions is determined using the gel clot LAL test (Pyrogent, Bio Whittaker), which is conducted according to the working instructions of the manufacturer, but with half the volumes (50 xcexcL) of sample, or diluted sample, and LAL reagent, whereby the endotoxin concentration is given in endotoxin units per ml and abbreviated in the following as EU/ml. The detection limit of the test kit is 0.06 EU/ml. Until measurement, the samples are stored in depyrogenated boron silicate tubes at 4-8xc2x0 C. for 48 hours at most. Samples stored longer are stored below xe2x88x9220xc2x0 C.
To determine the endotoxin retention capacity of hollow-fiber membranes, 50 capillaries of each of the membranes to be tested are integrated into test modules with a free membrane length of approximately 14 cm, so that an effective (interior) membrane surface of approximately 45 cm2 results. The test solution has an endotoxin concentration of lipopolysaccharide prepared from E. coli 055:B5 (List) in the range of 60-240 EU/ml in isotonic NaCl solution (B. Braun Melsungen) with addition of 1.5 mM CaCl2 and 0.75 mM MgCl2 (in part also in isotonic NaCl solution without CaCl2 and MgCl2). The test solution is filtered in dead-end mode with a hose pump from the intracapillary side to the extracapillary side, whereby the filtration rate is adjusted such that the pressure build-up calculated with the transmembranal flow measured for the respective membrane is on the order of 300 mbar during filtration. The filtration took place for 180 minutes, with sampling after 30, 60, 120, and 180 minutes. The endotoxin retention capacity is determined by comparing the endotoxin concentration in the test solution with that in the permeate.
To determine the endotoxin retention capacity of flat membranes, membrane discs with 47 mm diameter are punched out and integrated into depyrogenated stainless-steel filtration equipment (Sartorius; effective membrane surface 13 cm2). Test solutions with endotoxin concentrations of lipopolysaccharide prepared from E. coli 055:B5 (List) between 0.3 and 600 EU/ml are prepared with water (aqua ad iniect., Braun Melsungen) or with isotonic NaCl solution (Braun Melsungen) and placed in a 5 liter stainless steel container, whose outlet is connected via a PVC tube to the inlet of the filter holder. By applying 1.6 to 2 bar to the pressure reservoir, approx. 100 ml solution is filtered in each case through the membrane within 9 to 12 seconds and the filtrate collected in depyrogenated measurement cylinders. If endotoxin solutions of different concentrations are used during one experiment, they are filtered through the same filter starting with the lowest endotoxin concentration. The endotoxin retention capacity is determined by comparing the endotoxin concentration in the test solution with that in the filtrate.
The transmembranal flow TMF was measured as:
TMF=xcex94V/(xcex94txc2x7Axc2x7p) [ml/minxc2x7cm2xc2x7bar]
with distilled water at room temperature.
The measurement of the ultrafiltration rate and sieve coefficient is performed using the methods described in EP-A 96 915 007, incorporated herein by reference. The measurement of the maximum pore size is performed using the blow-point method described in EP-B 0 361 085, incorporated herein by reference. The invention will be described in more detail in the following examples, whereby the filtrations and endotoxin determinations are conducted using the previously described methods unless otherwise stated.